Editorial Article
Monday July 13, 2009
by Caitlin Smith
Imagine studying molecular interactions, even at the cellular level, without fluorescent labels or other molecular tags. You can do this with several types of label-free technology. Two obvious advantages of these techniques are that the label cannot interfere with binding or other molecular interactions, and that the technology is not toxic to cells—especially important for precious cultures such as primary or stem cells. Until recently, label-free technology has been used mainly in biochemical binding assays; now it is used for cell-based assays for such sought-after drug targets as G-protein-coupled receptors (GPCRs) and receptor tyrosine kinases.
“Being allowed to analyze receptors that are expressed in a natural environment at natural levels with the appropriate accessory proteins gives researchers a window into how these receptors function in an environment which is far more bio-relevant,” says David Yamane, senior director of drug discovery marketing at MDS Analytical Technologies. “The label-free technologies are particularly advantageous for Gi-coupled and Gs-coupled GPCRs, because they provide superior signal window, dynamic range, and reproducibility compared to typical labeled technologies. Additionally, label-free technologies enable enhanced study of complex pharmacological agents, such as allosteric modulators, partial agonists, and inverse agonists.”
Yamane also notes that a valuable asset of label-free technology is the “universality of the measurements produced. With label-free technologies, researchers can measure a wide range of targets using one platform with little additional assay development,” says Yamane. “Label-free technologies have been used repeatedly to measure a wide range of GPCRs and tyrosine kinase receptors, and are well-accepted for this purpose. In addition, they are being used increasingly to measure ion channels, adhesion molecules, and other critical targets. No labeled technology has this versatility. This flexibility allows researchers to conveniently screen a broad number and wide variety of targets with a single instrument platform, which helps standardize data to support more optimized lead compound selection.” Today, a myriad of technologies aim to accomplish these advantages, albeit with different approaches.
Using protein signals
Most therapeutic drugs evoke an antibody response, which can lead to harmful effects or reduced drug efficacy. Biacore™ (part of GE Healthcare) offers a label-free protein interaction analysis tool, the T100 Immunogenicity Package, to screen for anti-drug antibodies that interact with therapeutic drugs with rapid kinetics. The Package includes support for screening, conformation, and characterization of immune responses.
Using acoustical and electrical signals
TTP Labtech’s RAPid4® uses resonant acoustic profiling, in which molecules binding to a quartz crystal sensor chip are detected by a change in the crystal’s oscillating frequency. This technology has an advantage over others in that it can analyze crude samples or complex mixtures such as cell lysates, serum, and cell culture media. The RAPid4 can process four samples simultaneously, running about 350 samples per day.
MDS Analytical Technologies’ label-free technology is based on changes in impedance measured by electrodes in microplates. Their new CellKey®384 System offers high-throughput capacities with 384-well plates and incorporated fluidics. “All CellKey Systems enable researchers to dispense compounds into sample wells while simultaneously measuring the kinetic cellular responses,” says Yamane. “Since researchers do not have to shuttle sample plates from an offline pipettor to the CellKey System, they can measure 100% of the response, with no gaps in the measurement for sample addition. This feature, combined with the nature of impedance measurement, enables researchers to acquire robust quantitative and qualitative measurements from each response.” The CellKey System also allows the study of both adherent and non-adherent cells in the absence of artificial extracellular matrices. “This is particularly advantageous for studies of non-adherent cells where the use of coatings required by other analysis methods could alter native signaling pathways,” notes Yamane.
Focusing on specific cell types may yield more specific data to prioritize lead compounds in drug discovery, according to Yamane. “The CellKey® System enables researchers to study critical cell signaling receptors, such as GPCRs, in the natural environment, by providing the sensitivity to analyze endogenous targets,” he says. “With the CellKey® System, researchers can study both common cell lines and primary cell types. In fact, researchers are compiling a growing body of data for a range of primary cell types, including HUVECs, PBMCs, and neuronal cells.”
Using light signals
SRU Biosystems uses optical resonance in their BIND® technology based on BIND® readers and a range of biosensors. “Our biosensors contain an optical grating, so that when the biosensor is illuminated with multi-wavelength light, only a very narrow range of wavelengths is reflected,” says Steger. “The peak wavelength that is reflected changes when things bind to the biosensor surface.” Their systems are suitable for both biochemical and cell-based assays, and for lower (16-well) and higher (1536-well) throughputs. SRU is hoping to use their optical resonance technology for label-free cellular imaging and other applications in the area between high-throughput screening and high-content screening, according to Steger. “Many compound profiling programs are now using a combination of label-free systems: higher throughput systems such as SRU’s to narrow down the number of compounds (down to hundreds rather than thousands), followed by the detailed analysis on those few compounds using Biacore,” says Steger. “We have seen a huge increase in that need. The lower-throughput systems, such as the Biacore, will always have their place.” SRU is also working on applying their BIND® system to the study of ion channels, and high-content screening of stem cells.
Corning will also offer a new 1536-well format microplate in the fall for label-free research with their Corning Epic® Systems. Their technology is based on changes in the index of refraction off the sensor/plate. Like SRU’s, Corning’s system can perform both biochemical and cellular assays. “Most competing label-free technologies are capable [of performing] either biochemical or cell-based analysis in low to medium throughputs, while Epic is able to screen either target class at throughputs up to 40,000 wells in eight hours,” says Ron Verkleeren, business director for Corning Epic® Systems. “Moreover, Epic is unique because of the high sensitivity of our optical biosensors and temperature controlled environment, which enable robust assay results for even very challenging systems.”
Yamane says that the cellular responses currently used, despite their power, are lacking in specificity. “As with labeled technologies, researchers must have tools to tease apart and understand how compounds are acting on the cell system of interest,” he says. “Researchers are now expanding this set [of tools] to better meet the needs of their specific experimental objectives.”